1. Field of the Invention
The present invention relates to an autoradiographic process and kits for said process.
2. Description of the Prior Arts
There has been heretofore known a radiographic process termed "autoradiography" or "radioautography" comprising steps of: introducing a radioactively labeled substance into an organism; placing the organism or a part of tissue of the organism (that is, a sample or specimen) and a radiographic film such as a high sensitivity type X-ray film together in layers for a certain period of time to expose said film thereto; and obtaining the locational information on the radioactively labeled substance in said specimen from the resolved pattern of the film. The autoradiography has been utilized, for example, to investigate the pathway and state of metabolism, absorption, and excretion of the substance introduced in the organism in detail. Such autoradiography is described, for instance, in the following literature: Method in Biochemical Experiment, Volume 6, Method in Tracer Experiment I, 271-289, "8. Autoradiography" by Toru Sueyoshi & Akiyo Shigematsu (Tokyo Kagaku Dozin Ltd., 1977).
The autoradiography has been also utilized to obtain locational information on the radioactively labeled substances present on a medium containing radioactively labeled tissue of an organism and/or the radioactively labeled substances originating from an organism. For instance, there is known an autoradiography comprising steps of: labeling organism-originating biopolymers such as proteins or nucleic acids with a radioactive element; resolving the mixture of the radioactively labeled biopolymers, derivatives thereof, or cleavage products thereof on a gel support (medium) through a resolving process such as gel electrophoresis; placing the gel support and a high sensitivity X-ray film together in layers for a certain period of time to expose said film to the gel support, developing said film, obtaining the locational information of the radioactively labeled substances from the developed film, and then performing the identification of the polymeric substances, determination of molecular weight of the polymeric substances and isolation of the polymeric substances based on the obtained locational information.
Recently, the autoradiography has been effectively used especially for determining the base sequence of a nucleic acid such as DNA. Therefore, the autoradiography is thought to be a very useful means in the field of structural determination of polymeric substances originating from organisms.
Maxam-Gilbert method and Sanger-Coulson method are known as methods for sequencing DNA utilizing autoradiography. In these methods, the base sequence of DNA is determined by geniously utilizing the characteristic structure of DNA that DNA is in the form of a double helix structure, which consists of two chain molecules stabilized through hydrogen bonding between two bases on each chain molecule, and that the base, which comprises a part of constitutional unit of DNA, is limited to only four, which are adenine (A), guanine (G), cytosine (C), and thymine (T), and that the hydrogen bonding between each constituting base unit comprises only two combinations, namely, G-C and A-T.
For instance, Maxam-Gilbert method is performed by the procedure described below.
A group containing a radioactive isotope of phosphor (P) is attached to a chain molecule of DNA or a DNA fragment at one end to be sequenced to prepare a radioactively labeled substance, and then the radioactively labeled DNA molecule is specifically cleaved at the specific constitutional unit containing a certain base by a certain chemical reaction. This reaction is called a "base specific cleavage reaction". Then a mixture of numerous cleavage products of the DNA or DNA fragment, which is formed base-specifically by the above-mentioned procedure is resolved through gel electrophoresis to obtain a resolved pattern, in which numerous cleavage products are resolved depending on the molecular weight, which is approximately proportional to the length of molecule of the cleavage products, to form a band spectrum, or a ladder pattern (the bands are not visible) on the gel. The electrophoresed gel is subsequently placed in contact with a high sensitivity X-ray film for a long time at a low temperature, whereby the X-ray film is exposed to the resolved pattern, to cause the radiation from the respective bands containing the radioactively labeled cleavage products to form a latent image of the resolved pattern thereon. The X-ray film having the latent image thereon is developed to obtain a visible band spectrum consisting of a large number of bands which corresponds to the resolved pattern. Then the distance of the each band of the base-specifically cleaved product from the starting position of electrophoresis, which corresponds reversibly to the sequential position from the radioisotopically labeled terminal end of the DNA molecule is obtained from the developed film. Thereafter, by arranging the bands of the base specific cleavage products of four bases in accordance with the distance obtained by the above-mentioned procedure, the sequential position of each base from the radioisotopically labeled end of the chain molecules is read by referring to the applied based specific chemical reaction.
Maxam-Gilbert method summarized above is described in detail in the following literature: METHODS IN ENZYMOLOGY, VOL., 65, PART I (ACADEMIC PRESS, NEW YORK LONDON TORONTO SIDNEY SAN FRANCISCO, 1980)
Sanger-Coulson method also utilizes the specific structure of DNA and is employable for determining the sequence of bases in DNA by the use of DNA synthesis enzyme, gel electrophoresis, and the autoradiography techniques.
The characteristics and procedures of Sanger-Coulson method as well as those of the above-mentioned MaxamGilbert method are briefly described in the following literature: "Reading the genetic information in the original language. A surprising method for sequencing the bases of DNA" written in Japanese by Kin-ichiro Miura, Modern Chemistry, Sep. 1977, pp. 46-54 (Tokyo Kagaku Dozin Ltd., Japan).
As described above, the autoradiography is a very useful method for obtaining one or two dimensional information on the location of the radioactively labeled substances present in a sample such as tissue of an organism and a medium containing substances or tissues originating from an organism. Thus, the autoradiography is advantageously applicable, for instance, to the investigation of the pathway and state of metabolism, absorption, and excretion of a substance introduced in an organism, as well as to the determination of the structure of biopolymer.
Nevertheless, such useful autoradiography is not free from several drawbacks in the practical use.
In the first place, a long period of time and a complicated operations are required for performing the procedure of exposing a radiographic film such as a high sensitivity X-ray film to a radioactive element-containing sample such as the autoradiogram, an organism dosed with radioactively labeled substance or a portion of the tissue of the dosed organism through placing the sample and the film together in layers so as to visualize the position of the radioactive substance. More in detail, in the conventional autoradiography, the above-mentioned exposing procedure is performed at a low temperature (for instance, in the vicinity of 0.degree. C., or -70.degree. C. to -90.degree. C. in the course of the base-sequencing of a nucleic acid) for a long period of time (for instance, several days). The reasons why these conditions are involved are that a long exposure time is required to attain an appropriate exposure because generally a sample subjected to the autoradiography is not provided with high radioactivity, and also because the photosensitive silver salt of the radiographic film is chemically fogged by various substances contained in the sample when the film is kept at a relatively high temperature such as room temperature for a long period of time during the exposure, resulting in difficulty of obtaining an exposed image with high accuracy. Thus, the exposure ought to be carried out at a low temperature to depress the chemical fog. It can be considered that a radiographic film be more sensitized to mitigate the severe exposure conditions, but a radiographic film used in the conventional autoradiography is provided already with very high sensitivity and the satisfactory further enhancement in the sensitivity can not be expected if the sharpness of an image to be obtained is taken into consideration.
In the second place, the photosensitive silver salt of a radiographic film has a drawback that it is sensitive not only to the chemical irritation but also to physical impetus, and this drawback brings about difficulty in the autoradiographic procedure and decreases accuracy thereof. More in detail, since the exposing procedure is necessarily carried out keeping a radiographic film in contact with a sample, the radiographic film is generally handled with no protective cover during operations such as transferring and installing operations for the radiographic film. Accordingly, the radiographic film is likely be brought into contact with hands of the operator and tools in the handling, and the physical pressure arising from these contacts causes production of the physical fog on the radiographic film. Thus produced physical fog is also a cause of the decrease of accuracy in the autoradiography. For this reason, the handling of a radiographic film requires well-trained skill and a great caution to avoid the production of the physical fog on the radiographic film, and such careful handling required brings about increases the complexity in the autoradiographic procedure.
In the third place, certain natural radioactive substances contained in the sample in addition to the radioactively labeled substance take part in the exposure of the radiographic film because the exposure is carried out, as described above, for a long time in the conventional autoradiography. Thus, the influence of the natural radioactive substance further reduces the accuracy of locational information on the radioactively labeled substances, and this is another drawback. In order to remove the troublesome noise brought about by the natural radioactive substances, parallel experiments using control samples and a method for optimization of the exposure time have been employed, but these procedures include increased experimental runs for the parallel experiments and requires preliminary experiments to determine the preferable exposure time, and thus the drawback arising from the complicated procedures not avoidable as a whole.